Enzo Leone

A ribonuclease from human seminal plasma active on double -stranded RNA

A ribonuclease, active on single- and double-stranded RNAs, has been isolated from human seminal plasma 3-5 micrograms of enzyme were recovered per ml of seminal plasma, equivalent to 71% of total activity and a 2500-fold purification (measured with poly(A) X poly(U) as substrate) from the initial dialyzed material. Similar amounts of RNAase were found per g (wet weight) of human prostate, where the enzyme appears to be produced. Human seminal RNAase degrades poly(U) 3-times faster than poly(A) X poly(U), and poly(C) or viral single-stranded RNA about 10-times faster than poly(U). Degradation of poly(A) X poly(U), viral double-stranded RNA, and poly(A) by human seminal RNAase is 500-, 380- and 140-times more efficient, respectively, than by bovine RNAase A. The enzyme, a basic protein with maximum absorbance at 276 nm, occurs in two almost equivalent forms, one of which is glycosylated. Mr values of the glycosylated and non-glycosylated form are 21000 and 16000, respectively. The amino-acid composition of the RNAase is very similar to that of human pancreatic RNAase. The same is true for the carbohydrate content of its glycosylated form.

Ribonuclease BS-1: Sequence of two cyanogen bromide peptides

Summary When native RNAase BS-1 x is treated with cyanogen bromide, one peptide (CB2) is cleaved from the rest of the protein (CB1) and can be isolated by gel filtration. Peptide CB2 has been sequenced and shown to be the N-terminal peptide of the protein, strictly homologous to the peptide 1–13 of RNAase A. A second fragment (peptide PF2) can be separated by gel filtration after performic acid oxidation of the CB1 fragment. The sequence of this peptide is partially homologous to the 14–29 sequence of RNAase A. From the recovery values determined for peptides CB2 and PF2, it appears that each peptide is present in both chains of RNAase BS-l. These and previous findings are briefly discussed in terms of structure to function relationships.

Inhibition of ADP-Ribosylation Reaction by 2′, 5′-Oligoadenylates

Many experimental data have been obtained regarding the physiological functions of poly(ADP-ribosyl)ation reactions. DNA replication, cell differentiation, transformation of cells, and DNA repair are among the processes in which poly(ADP-ribosyl)-ation is thought to be involved [1–3]. Inhibitors of ADP-ribosyltransferase (ADPRT) have been very useful in the attempt to ascribe a physiological role to these reactions in various cellular functions. 3-Aminobenzamide and 3-methoxybenzamide have been shown to be powerful competitive inhibitors of ADPRT [4]. Some experimental data obtained with the use of these inhibitors clearly indicate that poly(ADP-ribosyl)ation reactions are indeed crucial for cell differentiation [3] and the DNA strand break rejoining process [5]. On the other hand, the use of inhibitors often meets inconveniences in that their effects are not always limited to the desired metabolic pathway [6]. Tanaka et al. [7] have recently reported that diadenosine tetraphosphate, a ligand of a subunit of DNA polymerase α, could act as a very efficient inhibitor of ADPRT, but further data concerning its involvement in ADP-ribosylation reactions have not been reported so far. Poly amines, such as spermine and spermidine, are also known to have inhibitory effects on ADP-ribosylation of histone H1 [8], although their usefulness as a specific inhibitor of ADPRT remains doubtful. In the light of this circumstance, we have tried to examine the effect of various 2′,5′-oligoadenylates (2′,5′A) on the activity of ADPRT.

Nucleic Acids, Histones and Spermiogenesis: The Poly(Adenosine Diphosphate Ribose)Polymerase System

The enzyme poly(adenosine diphosphate ribose)polymerase was discovered in chicken liver nuclei in 1966 (Chambon et al., 1) and thoroughly studied in a number of laboratories, where its main properties have been investigated; major contributions in this regard have been given by the groups of Hayaishi (2) and Sugimura (3). The enzyme has many peculiar properties.

Reversible inactivation of ribonucleases by ADPribosylation

The activity of purified bovine seminal RNAase and pancreatic RNAase A (EC 3.1.27.5) has been investigated following in vitro ADPribosylation in the presence of nuclear ADPribosyltransferase (EC 2.4.2.30) and NAD+ X ADPribosylation of these enzymes was correlated with a significant decrease in their activities. Approximately three residues of ADPribose were present per mol of enzyme. Removal of the bound ADPribose restored enzyme activity to near normal levels. Similar results were obtained with nuclei isolated from bull seminal vesicles as an endogenous source of seminal RNAase and nuclear ADPribosyltransferase. The findings suggest that in vitro ADPribosylation has a reversible inactivating effect on ribonucleases.

ADP-ribosylation of a specific protein from isolated intact bull testis nuclei

ADP-ribosylation of a specific basic protein has been investigated in isolated intact bull testis nuclei incubated with NAD+. The electrophoretic mobility, molecular weight and amino-acid composition of the purified bull testis specific protein are similar to those of rat testis protein. About 1-5% of the total radioactivity incorporated in the 20% acid-insoluble fraction was associated with testis protein and was identified as ADP-ribose.

Poly(ADP)-Ribosylation of Nuclear Proteins in the Mouse Testis

Poly(ADP-ribosylation) of nuclear proteins is one of a number of post-translational events which has been demonstrated in eukaryotic cells. The reaction is catalysed by poly(ADP-ribose) synthetase which transfers ADP-ribose from NAD to a suitable acceptor protein. Histone H1 and HMG proteins 1, 2, 14 and 17 have been reported as acceptors in a variety of tissues ranging from trout testis [1] to mammary carcinoma cells [2]. Poly(ADP-ribosylation) of HMG proteins is of particular interest because of the reported association of these proteins with actively transcribed genes [3]. Functionally, poly(ADP-ribosylation) has been implicated in a variety of regulatory events such as DNA synthesis [4], DNA excision repair [5, 1], gene expression [6] and cell differentiation [7].